Autophagy Inhibition Enhances Apoptosis Induced by Dioscin in Huh7 Cells
Figure 5
Autophagy inhibitors have effects on dioscin-induced cell death. Cells were treated with dioscin of 2.5 and 5 μM for 24 hours in the presence or absence of autophagy inhibitor 3-MA of 5 mM and then subjected to western blotting for LC3-II, beclin-1, cleaved PARP, caspase 3, Bcl-2, and β-actin (a). Subsequently, these treated cells were double stained with Annexin-V and PI and then analyzed by flow cytometry (b). For another inhibitor BafA1, cells were treated with an indicated concentration of dioscin for 24 hours in the presence or absence of 10 nM BafA1 and then subjected to MTT assay for cell viability. The expression of LC3-II formation was investigated by western blotting with β-actin being an internal control (c). These treated cells were double-stained with Annexin-V and PI and subsequently analyzed by flow cytometry (d). Results are shown as mean ± SD. *, dioscin versus BafA1 plus dioscin. Cells were treated with dioscin of 2.5 and 5 μM for 24 hours in the presence or absence of caspase inhibitor Z-VAD-FMK of 20 μM and then subjected to western blotting for LC3-II, beclin-1, PARP, caspase 3, Bcl-2, and β-actin (e). Subsequently, these treated cells were double stained with Annexin-V and PI and then analyzed by flow cytometry (f). Furthermore, cells were treated with various combinations for 24 hours, including 20 μM Z-VAD-FMK, 5 μM dioscin plus 20 μM Z-VAD-FMK, 5 mM 3-MA, 5 μM dioscin plus 5 mM 3-MA, 5 μM dioscin plus 20 μM Z-VAD-FMK and 5 mM 3-MA, and then subjected to MTT assay for cell viability (g). Results are shown as mean ± SD (g). ***, control versus dioscin; #, dioscin versus 3-MA plus dioscin or Z-VAD-FMK and 3-MA plus dioscin.
